Probably because of my background in biochemistry, the molecular mechanisms that allow our body to maintain homeostasis and ensure that all physiological functions work perfectly well have always fascinated me. Besides, I believe that understanding them also helps us get the big picture of the interconnection of the different physiological processes and enhance the effectiveness and rigour of the decision-making as performance nutritionists and training coaches. Not to mention its undeniable importance in finding effective treatments for diseases.
In this blog I want to talk about the glucose transporter GLUT4, focusing on the underlying mechanism of its regulation. GLUT4 plays a pivotal role in glucose homeostasis by enabling glucose uptake in response to insulin and exercise. Unmasking the molecular mechanisms of its regulation has special importance in understanding the molecular and physiological responses to exercise and finding more effective treatments for metabolic diseases such as diabetes mellitus.
Let me first put you into context before we dive in.
Carbohydrates have been shown to be crucial for the survival of human beings. Indeed, the brain is dependent on glucose – the form of carbohydrate that our body uses as energy source – as its main fuel. Also, during exercise, not only are carbohydrates the preferred fuel at moderate-to-high intensity, but their oxidation is a required process for fat metabolism during low-intensity exercise – fats burn in the flame of carbohydrates. These are just the most common examples of the key role that carbohydrates have in our life.
Fats burn in the flame of carbohydrates
The circulatory system, among other functions, is responsible for supplying the different organs of our body with the necessary amount of glucose for their metabolism. Blood glucose then has a critical role within the human physiology. Low circulating glucose levels – also called hypoglycaemia – can cause dizziness, cognitive dysfunction and even lead to coma.
Before undergoing hypoglycaemia, glucagon release is triggered, which makes the liver break down glycogen to glucose (glycogenolysis) or synthesize it de novo (gluconeogenesis) mostly from alanine and lactate and release it to the blood so that a drop in blood glucose levels is prevented. Also, when we eat, the carbohydrates ingested end up in the blood, which prevents hypoglycaemia as well.
When we eat, however, blood glucose levels raise over the normal physiological values – what we call hyperglycaemia. This can eventually lead to the development of diabetes and even cause a diabetic coma – or ketoacidosis – if mechanisms to bring them back down to normal values are not turned on.
When blood glucose levels go up, insulin is released and glucose uptake by different tissues throughout the body – such as the liver, the adipose tissue, the heart, and the skeletal muscle – is stimulated so that normoglycemia is maintained.
Apart from insulin, exercise is also a powerful stimulus for the skeletal muscle tissue to enhance glucose uptake from the blood, which aims to provide the muscle fiber with enough amount of substrate for oxidation so that the energy demands are met. Then, as you see, the glucose uptake by the skeletal muscle is regulated by a double-edged mechanism. Isn’t it interesting?
But the main character here is the GLUT4 transporter, and it is because of its characteristics – apart from the contractility of the muscle fibers – that this double-edged mechanism exists. If you reached this point, you most likely want to know why, what these characteristics are and how it happens. But don’t worry, you are about to find out!
Before we start, however, I want to point out that this is one of the topics that demonstrates the very little knowledge we have about human physiology and body functioning, and the whole lot of events, processes, molecules, etc. that remain unmasked by science. Such a complex organism is not easy to understand. But this is, in part, what makes human and exercise physiology such a great area of science!
Well, no more rambling. Let’s get to the substance now!
What is the GLUT4 transporter?
GLUT4, discovered in 1988, is one of the 13 facilitative glucose transport proteins that exist and is principally expressed in skeletal and cardiac muscles as well as adipocytes.
GLUT4 controls glucose transport into fat and muscle tissues in response to insulin and also into muscle during exercise.
Unlike the other 13 isoforms of the glucose transporters, GLUT4 characterizes by its sensitivity to insulin and muscle contraction. This leads to the fact that, contrary to most other cells in the body, where glucose transport is constitutively active, in muscle and fat cells it is rapidly upregulated in response to insulin, and in muscle tissue during exercise.
What makes the upregulation of glucose uptake possible?
The key of this upregulation property of glucose uptake by GLUT4 relays on the fact that, in a resting state, only a small percentage of the glucose transporter is located on the basal membrane while the most of it is located in intracellular compartments or endosomes that received the name of GLUT4 storage vesicles (GSVs).
It is well established that both insulin and exercise enhance glucose transport by mobilizing GLUT4 from the GSVs to the plasma membrane of the cell, a process called translocation. Obviously, it is at the surface of the cell where they can exert their function.
Since there is a constant turnover of GLUT4 from the plasma membrane to the GSVs and vice versa, the effects of insulin and exercise on glucose transport are believed to be due to an enhanced exocytosis of GSVs.
The molecular basis of GLUT4 vesicular trafficking, docking, and fusion is not completely understood. However, I want to give you some basics so you can get the big picture of how it works, even though you should bear in mind that it is an oversimplification.
There is good evidence that the process of docking and fusion of GSVs to the plasma membrane is regulated by a family of proteins called SNARE proteins. Vesicle (v-) SNARES are SNARE proteins located in the GLUT4 vesicles, and target (t-) SNARES are membrane proteins that are located at the cell membrane. When v-SNARES interact with the relevant t-SNARE, A SNARE complex is formed and the fusion of the vesicle with the plasma membrane is stimulated.
The relevant SNARE proteins that have so far been involved in docking and fusion of GSVs are VAMP2 (v-SNARE), sintaxin 4 (t-SNARE), and SNAP23 (t-SNARE).
We now know the ultimate result of insulin or exercise stimulation. Let’s now move on and get into the mechanisms by which this is achieved. Before that, however, I want to make clear that the exact mechanisms by which GLUT4 translocation is stimulated by insulin and exercise is not completely understood and many gaps in the process exist. Also, if you dive into the literature, you will find that some studies support a certain model whereas some others undermine it – most likely due to differences in the methodology used.
Here I will expose the most supported version of the model, while omitting the processes or intracellular signals that have been barely studied. Bear in mind that this can undergo some changes as further research into it is performed, although I will try to keep it as updated as possible over time.
Are you still there?
Regulation of GLUT4 translocation by insulin
As I already said in the introduction to this article, when blood glucose levels go over the normal range (79-110 mg/dL) insulin is released, which triggers GLUT4 translocation to the plasma membrane and hence an enhanced glucose uptake.
For insulin to create any change in any cell, it has first to be recognized by them. This means that insulin must bind an insulin-specific receptor exposed on the surface of target cells. It is well established that the insulin receptor (IR) is a tyrosine kinase. This means that upon activation it phosphorylates different substrates that are crucial for insulin to execute certain changes in the target cells. In this case, IRS1 is the substrate responsible for the waterfall of events that insulin leads to.
IRS1 is an adapter that aims to recruit other intracellular molecules to further transmit the signal, such as the phosphatidylinositol 3-kinase (PI3K), the next relevant molecule in the insulin signalling. You should know, and you probably know already, that the plasma membrane is made of different types of lipids. One of these lipids is the phosphatidylinositol-4,5-bisphosphate (PIP2). PI3K, after binding to IRS1, phosphorylates this lipid leading to increasedphosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. This last step leads to the activation of PDK1, which then phosphorylates and activates Akt, which in turn phosphorylates different other substrates.
Two of the substrates that Akt phosphorylates are two RabGAPs – GAP stands for GTPase-activating protein – related to the GLUT4 translocation pathway, TBC1D4 (also known as AS160) and TBC1D1 proteins. RabGAPs are proteins that stimulate the GTPase activity of a subfamily of small G proteins named Rab, which are active when bound to GTP. Therefore, RabGAPs indirectly induce the inactivation of Rab proteins by making them hydrolyse GTP to GDP. In turn, when bound to GTP thus active, Rab proteins are believed to induce exocytosis of GSVs and hence GLUT4 translocation to the plasma membrane.
It is important to highlight that TBC1D4 and TBC1D1 are constitutively active, and phosphorylation by Akt induces their sequestration by binding to the protein 14-3-3 and, therefore, their inactivation.
I know it is not an easy topic and you are probably struggling to make the entire pathway in your head. Let me wrap it up for you. We’ve got that when insulin is released and binds to the IRs on the surface of target cells, a series of phosphorylation where IRS1, PI3K, and PKD1 lead to Akt phosphorylation and activation. TBC1D4 and TBC1D1, that where active and thus inhibiting Rab, then are phosphorylated by Akt and hence sequestered by 14-3-3, which prevents them from inactivating Rab. Then Rab can be activated by binding GTP and stimulate the fusion of GSVs to the plasma membrane in order to favour GLUT4 translocation, which ultimately enhances glucose uptake.
It is pretty well-established that PI3K activates Rac1, a member of another family of small G proteins called Rho-GTPases. Rac1 which is involved in the cytoskeleton dynamics. When active, Rac1 reorganizes the actin filaments beneath the membrane, a step required for GSVs exocytosis and GLUT4 translocation.
You now know how is the signalling mechanisms that induces GLUT4 translocation to the plasma membrane in response to insulin. Let’s now look at the one caused by exercise.
Regulation of GLUT4 translocation by exercise
After finding out about the previous signalling pathway you may be scared of the one following exercise. I understand it. But don’t worry because they both share a big part of it. The signalling pathway generated by both stimuli converges at the phosphorylation of TBC1D4 and TBC1D1. So you already know the final steps of this pathway. But… how does it begin? Let’s look into it!
We know that the main intracellular changes caused by exercise are an increased concentration of calcium and an increased concentration of AMP with the concomitant decrease in ATP concentration. Let’s go part by part.
As already addressed in a previous blog about AMPK, the increased AMP:ATP ratio caused during exercise induces AMPK activation by facilitating its phosphorylation by upstream kinases. When active, AMPK is believed to be one of the enzymes responsible for the phosphorylation and inactivation of TBC1D4 and TBC1D1.
On the other side, the accumulation of intracellular calcium activates both CaMKK2 and CaMKII. The former one, as thoroughly reviewed in the blog about AMPK, has been shown to be one of the upstream kinases of AMPK, thus stimulating sequestering and inactivation of TBC1D4 and TBC1D1. CaMKII, instead, is believed to directly phosphorylate and inactivate TBC1D4 and TBC1D1.
The rest of the pathway follows as described in the insulin-induced GLUT4 translocation mechanism. In short, contraction during exercise causes an increase in both the AMP:ATP ratio and intracellular calcium. On one side, the increase in AMP:ATP ratio promotes activation of AMPK which in turn phosphorylates and inactivates TBC1D4 and TBC1D1. On the other side, intracellular calcium stimulates AMPK activation through phosphorylation of CaMKK2 and TBC1D4 and TBC1D1 inactivation through phosphorylation by CaMKII. By either way, TBC1D4 and TBC1D1 binding to 14-3-3 is stimulated and they become inactive. This allows Rab to stay active and promote GLUT4 translocation to the plasma membrane, ultimately enhancing glucose uptake.
As it happens with insulin stimulation, Rac1 is also believed to become active with exercise, leading to a reorganization of the cytoskeleton that enables GSVs exocytosis.
GLUT4 controls glucose transport into the different tissues where it is expressed – skeletal and cardiac muscles as well as adipose tissue – in response to insulin and, in muscle tissue, exercise. The most characteristic trait of this transporter, and key in its regulation, is that most of it is localized within intracellular compartments and only a few can be found at the plasma membrane at rest. When the cell is stimulated with insulin or exercise, the transporters are translocated to the sarcolemma, which leads to an enhanced glucose uptake.
One of the take home messages of this article would definitely be that science is still far from completely understanding human physiology. The regulatory mechanism of GLUT4 translocation by insulin and exercise has been of great interest among the scientific community since the 1990s, yet the exact mechanism by which it occurs is not well understood.
However, its comprehension would mean another step towards the human and exercise physiology understanding, and would definitely signify a progress in finding new treatments for metabolic diseases such as diabetes miellitus.
I hope you could follow this blog and process such technical information. As always, do not hesitate to contact me for discussing anything you didn’t understand or something you want to know more about. See you in the next blog!
- Klip, Amira, Timothy E. McGraw, and David E. James. 2019. “Thirty Sweet Years of GLUT4.” Journal of Biological Chemistry 294 (30): 11369–81.
- Mul, Joram D., Kristin I. Stanford, Michael F. Hirshman, and Laurie J. Goodyear. 2015. Exercise and Regulation of Carbohydrate Metabolism. Progress in Molecular Biology and Translational Science. 1st ed. Vol. 135. Elsevier Inc.
- Richter, Erik A., and Mark Hargreaves. 2013. “Exercise, GLUT4, and Skeletal Muscle Glucose Uptake.” Physiological Reviews 93 (3): 993–1017.